Palmitoylation Assay
Palmitoylation Assay - Related Videos
05:45
Assessing the Palmitoylation State of Cell Membrane Proteins from Mouse Brain Tissue
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2025
The video demonstrates the analysis of the palmitoylation state of cell membrane proteins isolated from mouse brain tissue. The process involves exposing the non-palmitoylated cysteines and blocking them, then using a polymer that binds to the palmitoylated cysteines, aiding in assessing the palmitoylation state of the isolated proteins.
08:28
Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins
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Cited by 7 •
2020
Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.
16:33
Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)
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Cited by 108 •
2013
The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.
08:12
The TUNEL Assay
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2023
One of the hallmarks of apoptosis is the nuclear DNA fragmentation by nucleases. These enzymes are activated by caspases, the family of proteins that execute the cell death program. TUNEL assay is a method that takes advantage of this feature to detect apoptotic cells. In this assay, an enzyme called terminal deoxynucleotidyl transferase catalyzes the addition of dUTP nucleotides to the free 3’ ends of fragmented DNA. By using dUTPs that are labeled with chemical tags that can produce...
09:12
Aortic Ring Assay
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Cited by 42 •
2009
Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.
FAQs
Hydroxylamine selectively removes palmitate linked through thioester bonds, exposing cysteine thiols that were previously modified. Newly available thiols can then be labeled, allowing the assay to distinguish palmitoylated cysteines from unmodified ones. This chemical sequence provides the basis for estimating modification levels rather than simply detecting total protein abundance.
Because palmitoylation can be reversed, it can dynamically alter a protein’s behavior rather than permanently changing its structure. Changes in modification status may influence membrane association, protein stability, trafficking, and signaling. A palmitoylation assay therefore helps connect a biochemical modification state with broader changes in cellular protein regulation.
Chemical assays exploit the difference between palmitoylated and unmodified thiols, typically by removing palmitate and labeling the cysteines that become exposed. Metabolic labeling instead tracks palmitate incorporation in cells, and click chemistry can support detection of the incorporated label. These strategies provide complementary ways to examine modification in biochemical or cellular settings.
A hydroxylamine-based workflow first treats the sample to remove thioester-linked palmitate. The cysteine thiols exposed by that treatment are then labeled, producing a signal associated with previously palmitoylated sites. Interpreting the labeling pattern or level helps identify modified proteins and estimate how extensively palmitoylation occurred.
The assay can indicate whether a protein is palmitoylated and can help estimate the level of modification. Those measurements support investigations of how palmitoylation relates to membrane association, stability, trafficking, or signaling. When applied across cellular pathways, the results can reveal post-translational regulation rather than merely documenting protein presence.
Metabolic labeling is useful when the research question concerns palmitate incorporation in cells rather than only the chemical state of an isolated sample. Coupling incorporation with click chemistry enables researchers to track the labeled palmitate signal and examine palmitoylation within cellular pathways, including processes relevant to signaling and disease-related regulation.